Tumour budding, poorly differentiated clusters, and T-cell response in colorectal cancer

Abstract

Background: Tumour budding and poorly differentiated clusters (PDC) represent forms of tumour invasion. We hypothesised that T-cell densities (reflecting adaptive anti-tumour immunity) might be inversely associated with tumour budding and PDC in colorectal carcinoma.

Methods: Utilising 915 colon and rectal carcinomas in two U.S.-wide prospective cohort studies, and multiplex immunofluorescence combined with machine learning algorithms, we assessed CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 co-expression patterns in lymphocytes. Tumour budding and PDC at invasive fronts were quantified by digital pathology and image analysis using the International tumour Budding Consensus Conference criteria. Using covariate data of 4,420 incident colorectal cancer cases, inverse probability weighting (IPW) was integrated with multivariable logistic regression analysis that assessed the association of T-cell subset densities with tumour budding and PDC while adjusting for selection bias due to tissue availability and potential confounders, including microsatellite instability status.

Findings: Tumour budding counts were inversely associated with density of CD3⁺CD8⁺ [lowest vs. highest: multivariable odds ratio (OR), 0.50; 95% confidence interval (CI), 0.35-0.70; P_trend < 0.001] and CD3⁺CD8⁺CD45RO⁺ cells (lowest vs. highest: multivariable OR, 0.44; 95% CI, 0.31-0.63; P_trend < 0.001) in tumour epithelial region. Tumour budding levels were associated with higher colorectal cancer-specific mortality (multivariable hazard ratio, 2.13; 95% CI, 1.57-2.89; P_trend < 0.001) in Cox regression analysis. There were no significant associations of PDC with T-cell subsets.

Interpretation: Tumour epithelial naïve and memory cytotoxic T cell densities are inversely associated with tumour budding at invasive fronts, suggesting that cytotoxic anti-tumour immunity suppresses tumour microinvasion.

Keywords: adenocarcinoma; artificial intelligence; clinical outcomes; epithelial mesenchymal transition; host-tumour interaction; molecular pathological epidemiology.

Fig. 1

Quantification of tumour budding and poorly differentiated clusters (PDCs) using International tumour Budding Consensus Conference (ITBCC) criteria and the densities of specific T-cell subsets using multiplex immunofluorescence. (a)-(b) Evaluation of tumour budding and PDCs using haematoxylin and eosin-stained sections. (a) Whole slide images were scanned at medium power to identify the most intensive areas of budding and PDCs. (b) The number of tumour buds (black arrowheads) and PDCs (yellow arrow head)/20x microscope field (0.785 mm²) were counted. (c)-(f) Evaluation of the densities and location of T cells with multiplex immunofluorescence. Machine learning-based image processing (c), included tissue category classification (d), cell segmentation (e), and cell phenotyping (f) to identify different T-cell subsets in intraepithelial and stromal regions. Abbreviations: ITBCC, International Tumour Budding Consensus Conference; PDC, Poorly differentiated clusters.

Fig. 2

Inverse probability weighting (IPW)-adjusted Kaplan-Meier curves of colorectal cancer-specific and overall survival according to tumour budding at invasive front. The P values were calculated using the weighted log-rank test for trend (two-sided). The table (bottom) shows the number of patients who remained alive and at risk of death at each time point after the diagnosis of colorectal cancer. .